A high resolution and mass accurate Orbitrap Velos hybrid FT mass spectrometer system is required to help uncover novel targets for smart drugs. Targeted drug therapies offer advantages over systemic chemotherapies since targeting specific proteins/genes minimizes side effects. Mass spectrometry based proteomics is well-suited to elucidate defective cancer cell signaling pathways with high sensitivity, throughput and specificity. We will perform both identification and quantification of peptides/proteins and modified peptides/proteins from cell lines and tumor tissues using stable isotope labeling (e.g., SILAC, iTRAQ, TMT) and label-free strategies (e.g., spectral counting, MS/MS average TIC peak area integration, MS peak profiling) through discovery and targeted experiments. In addition, protein-protein interaction (PPI) networks will be established in cancer signaling pathways. Data will be processed and analyzed using commercially available and freely available software (Sequest via Protein Discoverer, Mascot Browser, Scaffold, Scaffold PTM, MaxQuant and Cytoscape) and in-house developed software tools (PTM Quantifier and NakedQuant). Compared to the previous generation LTQ Orbitrap, the Velos Orbitrap represents a 2-fold improvement in scan speed and 3-fold sensitivity improvement resulting in a 2-fold improvement in depth of peptide and protein coverage. The mass spectrometer will be coupled to a splitless nanoflow HPLC in microcapillary LC/MS/MS mode for optimal sensitivity for cancer research. The Velos Orbitrap system will not be allowed open access and operated by trained professionals only. Service contracts, salaries, and supplies will be funded through a guaranteed operating account through BIDMC and will be recaptured through charge-backs from the NIH funded project grants. The Orbitrap Velos will be primarily shared among seven major users and three minor users in addition to being part of the fully equipped mass spectrometry core facility at Beth Israel Deaconess Medical Center (BIDMC) and accessible by additional PIs within the Longwood medical area subject to availability. Descriptions for five main projects that will utilize the technology are the (1) Evaluation of the functional role of the phosphoinositide-3-kinase (PI3K) complex contributing to AKT activation, (2) Dissection of the protein-protein interact-ome of the insulin signaling pathway in drosophila cells, (3) Quantification of protein components of the ribosome in disease models involving anemia, (4) Elucidation of the functional role of the tuberous sclerosis complex (TSC) tumor suppressors (TSC1 and TSC2) effects on the TORC complexes and (5) Proteomics characterization of Alzheimer's Disease brain plaques. We anticipate that the proposed experiments will uncover several druggable protein targets and eventually lead to one or more clinical trials. PUBLIC HEALTH RELEVANCE: A high resolution Thermo Scientific Orbitrap Velos mass spectrometer coupled to a splitless nano-HPLC will be used to discover new smart drug targets that are needed in diseases such as cancer to reduce side effects and efficiently attack and kill cancer cells based on the cell's defective pathways. Mass spectrometry based proteomics is ideal for elucidating the functional role of protein signaling pathways in diseased cells through quantitative and qualitative experimentation.